The invention relates to the oral delivery of peptide and protein pharmaceuticals using the vitamin B.sub.12 (VB.sub.12) uptake system. More particularly the invention relates to the amplification of the uptake system using polymers.
The oral route of administration of peptides such as LHRH and its analogues, or proteins such as Granulocyte Colony Stimulating Factor (GCSF), Erythropoietin (EPO) and insulin, as pharmaceuticals in the treatment of systemic conditions has in the past met with little success. In general, the amount of peptide required for successful oral administration has been 100 to 1000 times the dose required for parenteral delivery, thus making the administration of these agents via this route prohibitively expensive. There are two fundamental reasons for the lack of success. Firstly, the intestinal milieu has a high degree of proteolytic activity, which rapidly degrades most peptides. Secondly, while there are well defined uptake mechanisms for individual amino acids and di-peptides, there is no general mechanism for polypeptides to be transported across the membrane of the mucosal epithelium into the circulation. Rather, this membrane is designed as a general barrier prohibiting the uptake of the numerous foreign proteins encountered in this environment. Thus, although a peptide may be modified to withstand the enzymatic barrage encountered in the intestine, such modification is of little value if the peptide cannot subsequently cross the mucosal barrier and enter the systemic circulation.
Recent work by the current inventor, which is described in PCT Patent Application PCT/AU86/00299 (WO87/02251), has however provided a method to overcome the mucosal barrier. This method takes advantage of the natural intrinsic factor (IF) mediated uptake mechanism for vitamin B.sub.12 (VB.sub.12). VB.sub.12 is a naturally occurring dietary molecule which is actively taken up from the intestine. During this process it first binds to intrinsic factor (IF) in the upper small intestine. The [VB.sub.12 -IF] complex passes down the small intestine and binds to an IF receptor located on the surface of the ileal epithelium. The whole [VB.sub.12 -IF-Receptor] complex is then internalized by receptor-mediated endocytosis and some time later the VB.sub.12 appears in serum.
PCT Application PCT/AU86/00299 (WO87/02251) describes methods to modify chemically VB.sub.12 to provide suitable functional groups for conjugation of the VB.sub.12 to various drugs and peptide/protein pharmaceuticals. When the [VB.sub.12 -pharmaceutical] complex is administered orally it is possible to utilise the natural IF-mediated VB.sub.12 -uptake system to deliver the pharmaceutical to the circulation.
One major limitation to this general VB.sub.12 uptake mechanism is that the dose of pharmaceutical which can be delivered per feed to the recipient is low. The dose is directly proportional to the amount of VB.sub.12 which can be taken up per feed. Thus, in mice and rats it is only possible to deliver around 20-40 pMoles of pharmaceutical per dose, while in humans the quantity of pharmaceutical which can be delivered is approximately 1 nMole. While this level of uptake is sufficient to deliver pharmaceutically active doses of some substances, such as LHRH agonists, calcitonin and EPO, it is not sufficient to deliver proteins such as GCSF and insulin at quantities large enough to have a pharmacological effect. It would therefore be desirable to amplify the uptake capacity of the VB.sub.12 transport system, by at least 10 fold.
Amplification can occur by the linkage of the pharmaceutical to a polymer backbone to which a small number of VB.sub.12 molecules are linked, either subsequently, previously or at the same time. Preferably the linkage to the polymer, or the polymer to which the pharmaceutical is linked, should be biodegradable.
Potentially biodegradable polymers include dextran and its derivatives and amino acid polymers such polylysine, or poly-(glutamic acid). Non-biodegradable polymers include poly[N-(2-hydroxypropyl)-methacrylamide], to which is attached biodegradable side chains such as those containing ester linkages, or amino acid sequences cleavable within lysosomal vacuoles, ie, Gly-Phe-Leu-Gly (SEQ ID NO:1)(Rihova, B. and J. Kopecek. 1985 Biological properties of targetable poly[[N-(2-hydroxypropyl)-methacrylamide]-antibody complexes. J. Control Rel., 2:289-310). Other amino acid spacers cleavable by intracellular proteases include Gly-Phe-Ala; Gly-Phe-Ala-Gly; (SEQ ID NO:2) Gly-Phe-Tyr-Ala; (SEQ ID NO:3) and Gly-Phe-Tyr-Ala-Ala (SEQ ID NO:4) (Rejmanova, P., Obereigner, B., Kopecek, J. 1981 Makromol. Chem. 182: 1899-1915).
Polymers with pendant groups linked to the backbone via spacers containing aromatic diazo bonds are known. Many of these polymers have been designed specifically to release the pendant side groups following cleavage of the diazo bond by azo-reductases released by bacteria in the colon. These polymers however have proven to be unsuitable for delivering drugs systemically following oral administration, because these polymer-drug complexes are not absorbed intact from the intestine, but rather deliver their drug to the colon, following cleavage of the diazo bond by colonic enzymes. Small amounts of the released drug may eventually reach the circulation, but the amount which reaches the circulation has been found to be of little practical value.
Polymers to which are conjugated various cytotoxic drugs are also known. These polymers have been targeted to cancer cells using specific antibodies or sugar moieties. Once the drug-polymer has reached its target tissue the complex is endocytosed by the target cell and the pendant drug is released by the action of lysosomal enzymes, or by cleavage of a disulfide linked drug by intracellular glutathione. Oral delivery of such complexes has not, however, resulted in significant uptake of the drug-polymer complex from the intestinal lumen into the circulation.